Monoclonal antibody drugs have made significant progress in recent 15 years and have become driving force in pharmaceutical industry. Since 1996, about 30 monoclonal antibody drugs in total have been approved, wherein the annual sales for nine drugs reached over 1 billion US dollars. In 2010, the overall sales of monoclonal antibody drugs were over 30 billion US dollars and the annual rate of growth was over 10%. The monoclonal antibody only inhibits a single target due to the high specificity against the target thereof. However, it may be necessary to inhibit multiple targets/signal pathways to avoid a compensatory effect for tumors, autoimmune diseases, and other diseases. For viral infection, due to the high mutation rate of viruses, in general, it is necessary to inhibit multiple antigenic sites in order to prevent escape. There are several alternative solutions. One is to use polyclonal antibodies, or to obtain a heterodimer, e.g. a bispecific antibody, by modifying Fc fragments of antibodies. Another solution is to use an antibody mixture for treatment, wherein the antibody mixture comprises two or more antibodies against different epitopes on the same target, or against different targets.
U.S. Pat. No. 7,262,028 discloses a method for producing a bivalent antibody or a mixture of bivalent antibodies from a single host cell clone by expression of one light chain and different heavy chains, and also provides a method for producing a combination of antibodies which can be screened for the usefulness in various applications.
WO/2010/084197 describes a method for producing a mixture comprising two or more different antibodies from a single host cell clone. In one embodiment, a mixture of different monovalent antibodies is produced. In another embodiment, a mixture of monovalent and bivalent antibodies is produced. In the method, homodimers are stabilized by virtue of the natural exchange phenomenon between two Fab arms of IgG4, wherein some residues of the hinge region and CH3 domain which caused the phenomenon are changed. However, the patent does not mention whether the problem of existence of heterodimers is completely solved.
U.S. Pat. Nos. 5,789,208 and 6,335,163 described a method for expressing a library of polyclonal antibodies, wherein a library of polyclonal Fab fragments was expressed on a phage display vector, and then screened for the reactivity to antigens. The selected combinations of variable region genes of heavy chains and light chains are transferred in a linked pairing way into an eukaryotic expression vector comprising constant region genes so as to obtain a sub-library of complete polyclonal antibodies. After the sub-library is transfected into myeloma cells, stable clones would produce antibodies which can be mixed to obtain a mixture of monoclonal antibodies. By using the method, although it is theoretically possible to directly obtain polyclonal antibodies from one recombination production process by culturing a group of mixed transfected cells, there may be potential problems in terms of the stability of group of the mixed cells and thus the consistency of the produced polyclonal antibodies. In a pharmaceutically acceptable large-scale (industrial) production method, it is an arduous task to control different cells in a whole group. For example, the properties such as the growth rate of cells and the production rate of antibodies should be kept stable for all single clones in the non-clonal group, so that the ratio of the antibodies in the mixture of polyclonal antibodies can be kept constant. Therefore, although the production for mixed antibodies may have been realized in the art, there are still no ameanable solutions which are economically and practically sounding for large scale manufacturing.
Recently, Merck and Symphogen A/S company from Denmark signed an exclusive worldwide license agreement for Sym004. Sym004 is a novel antibody mixture which is now being developed and targets the epithelial growth factor receptor (EGFR).
Sym004 consists of two antibodies, can block ligand binding, receptor activation and downstream signaling, and is also considered to elicit removal of the EGFR receptors from the cancer cell surface by inducing EGFR internalization and degradation. Sym004 is currently being evaluated in a Phase I/II trial for the treatment of patients with advanced wild-type KRAS metastatic colorectal cancer (mCRC) who have previously progressed on treatment with standard chemotherapy and a commercially available anti-EGFR monoclonal antibody. In addition, a Phase II trial in patients with squamous cell carcinoma of the head and neck (SCCHN) who have failed anti-EGFR-based therapy is currently ongoing.
The antibody mixture technology of Symphogen A/S company involves the following: firstly obtaining a plurality of antibodies against the same target by an antibody screening platform, then performing molecular construction for each antibody, culturing cells in shake-flask, and mixing the cells, and culturing the cell mixture in a way of gradual amplification culture, then performing a optimized purification to obtain the final product. However, the method still involves the problems caused by unstable cell growth rate and antibody production rate, since recombinant host cells are used in the method for producing a mixture of various homodimers. Because a single antibody is expressed in a single cell in the method, the method does not involve the problem of heterodimers.
In all means, it would be a more ideal for producing a protein or antibody mixture, If two or more proteins or antibodies can be produced in single recombinant cell clone,